FIGURE 2.
MYH is active in HCT116 extracts and addresses GO/A but not AO/G or AO/C mispairs. A, nicking assay. The closed-circular homoduplex or the GO/A, AO/G, or AO/C substrates were incubated with recombinant, purified MYH-GST and APE1 for the indicated times and analyzed by agarose gel electrophoresis. The mobility of the closed-circular (cc), open-circular (oc), and linear (l) DNA molecules is indicated on the left. The quantification shows the percentage of open-circular substrate compared with the total amount of DNA (open-circular + closed-circular). Only the GO/A substrate was cleaved with appreciable efficiency. B, schematic representation of the MYH-dependent nucleotide incorporation assay. Short patch BER should replace the mispaired A with a radiolabeled C (asterisk). This should result in detectable radioactivity in band a of the NotI/BsaI digest. C, BER-dependent incorporation assay. The supercoiled homoduplex (lanes 1–3), GO/A (lanes 4–6), and AO/G (lanes 7–9) substrates were incubated with HCT116 extracts for the indicated time points. The reactions were supplemented with [α-32P]dCTP. As visualized in the autoradiograph, [32P]dCMP incorporation into fragment a of a NotI/BsaI-digested phagemid, indicative of short patch repair, was detected above background only in the GO/A substrate. D, the supercoiled homoduplex (lanes 1–3) and AO/C (lanes 4–6) substrates were incubated with the extracts for the indicated time points in reactions supplemented with [α-32P]dGTP. Only low levels of radioactive nucleotide were incorporated into fragment a of a NotI/BsaI-digested AO/C phagemid, indicative of inefficient BER. M, molecular size marker.